Mouse anti tuj1

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100/PBS and blocked with 5% skim milk in TBS-Tween20 (TBS-T) buffer. All antibodies were diluted in 5% skim milk/TBS-T. For neuronal markers analysis, following antibodies were used: mouse anti-MAP2 (Sigma; 1:2,000 dilution), mouse anti-Neurofilament H (Smi-32; 1:2,000), mouse anti-neuronal nuclei (NeuN; 1:500), rabbit anti-Synapsin 1 (Syn1; 1:500), rabbit anti-MAP2 (all Millipore; 1:2,000), mouse anti-Tuj1 (Cell Signaling, 1:2,000), rabbit anti-postsynaptic density protein 95 (PSD95; 1:250), rabbit anti-Drebrin (both Abcam; 1:500), rabbit anti-pan voltage-gated Na2 + channels (pan Nav; Alomone; 1:250), anti-vesicular glutamate transporter 1 (vGlut1; Synaptic Systems; 1:500), and goat polyclonal anti-PTB1 (Abcam; 1:200). Rabbit antibodies against c9RANT products, anti-poly(GP), anti-poly(GA), anti-poly(GR), anti-poly(PA), and anti-poly(PR), were generated as described previously (Gendron et al., 2013 (link); Mann et al., 2013 (link)) and used at 1:1,000 dilutions. Secondary fluorescent antibodies (Invitrogen) were used at 1:1000 dilutions. Nuclei were stained with Hoechst 33258 (Invitrogen). Confocal microscopy was performed using Zeiss LSM 510 microscope.

For immunofluorescence staining of NSC/NPCs, neurospheres were moved using tips to seed on Matrigel-coated coverslips for 20 min before detection, and cells on coverslips were fixed with 4% PFA for 15 min and blocked with 5% bovine serum albumin/0.3% Triton X-100 for 1 h at room temperature. Cells were then incubated with primary antibodies rabbit anti-Sox2 (1:400, Cell Signaling Technology, catalog no. 23064), mouse anti-Nestin (1:200, Millipore, catalog no. MAB353), rabbit anti-BrdU (1:100, Abcam, catalog no. ab152095), or mouse anti-Tuj1 (1:200, Cell Signaling Technology, catalog no. 4466) overnight at 4°C and with secondary antibodies anti-rabbit Alexa Fluor 488 (1:200, Invitrogen, catalog no. A-11008), anti-mouse Alexa Fluor 594 (1:200, Invitrogen, catalog no. A-11005), or anti-mouse Alexa Fluor 488 (1:200, Invitrogen, catalog no. A-11001) for 2 h at room temperature before counterstaining with 4',6-diamidino-2-phenylindole. The BrdU/Nestin and Tuj1 positive cells were counted in each group.